Noninvasive fractal biomarker of clock neurotransmitter disturbance in humans with dementia

Human motor activity has a robust, intrinsic fractal structure with similar patterns from minutes to hours. The fractal activity patterns appear to be physiologically important because the patterns persist under different environmental conditions but are significantly altered/reduced with aging and Alzheimer's disease (AD). Here, we report that dementia patients, known to have disrupted circadian rhythmicity, also have disrupted fractal activity patterns and that the disruption is more pronounced in patients with more amyloid plaques (a marker of AD severity). Moreover, the degree of fractal activity disruption is strongly associated with vasopressinergic and neurotensinergic neurons (two major circadian neurotransmitters) in postmortem suprachiasmatic nucleus (SCN), and can better predict changes of the two neurotransmitters than traditional circadian measures. These findings suggest that the SCN impacts human activity regulation at multiple time scales and that disrupted fractal activity may serve as a non-invasive biomarker of SCN neurodegeneration in dementia

Targeting the choroid plexus-CSF-brain nexus using peptides identified by phage display.

Drug delivery to the central nervous system requires the use of specific portals to enable drug entry into the brain and, as such, there is a growing need to identify processes that can enable drug transfer across both blood-brain and blood-cerebrospinal fluid barriers. Phage display is a powerful combinatorial technique that identifies specific peptides that can confer new activities to inactive particles. Identification of these peptides is directly dependent on the specific screening strategies used for their selection and retrieval. This chapter describes three selection strategies, which can be used to identify peptides that target the choroid plexus (CP) directly or for drug translocation across the CP and into cerebrospinal fluid

Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system

Background: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS.Methods: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity.Results: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependant binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles.Conclusion: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain

Blood-Cerebrospinal Fluid Barrier Gradients in Mild Cognitive Impairment and Alzheimer's Disease: Relationship to Inflammatory Cytokines and Chemokines

Background: The pathophysiology underlying altered blood-cerebrospinal fluid barrier (BCSFB) function in Alzheimer's disease (AD) is unknown but may relate to endothelial cell activation and cytokine mediated inflammation.Methods: Cerebrospinal fluid (CSF) and peripheral blood were concurrently collected from cognitively healthy controls (N = 21) and patients with mild cognitive impairment (MCI) (N = 8) or AD (N = 11). The paired serum and CSF samples were assayed for a panel of cytokines, chemokines, and related trophic factors using multiplex ELISAs. Dominance analysis models were conducted to determine the relative importance of the inflammatory factors in relationship to BCSFB permeability, as measured by CSF/serum ratios for urea, creatinine, and albumin.Results: BCSFB disruption to urea, a small molecule distributed by passive diffusion, had a full model coefficient of determination (r2) = 0.35, and large standardized dominance weights (>0.1) for monocyte chemoattractant protein-1, interleukin (IL)-15, IL-1rα, and IL-2 in serum. BCSFB disruption to creatinine, a larger molecule governed by active transport, had a full model r2 = 0.78, and large standardized dominance weights for monocyte inhibitor protein-1b in CSF and tumor necrosis factor-α in serum. BCSFB disruption to albumin, a much larger molecule, had a full model r2 = 0.62, and large standardized dominance weights for IL-17a, interferon-gamma, IL-2, and VEGF in CSF, as well IL-4 in serum.Conclusions: Inflammatory proteins have been widely documented in the AD brain. The results of the current study suggest that changes in BCSFB function resulting in altered permeability and transport are related to expression of specific inflammatory proteins, and that the shifting distribution of these proteins from serum to CSF in AD and MCI is correlated with more severe perturbations in BCSFB function

Ecrg4 expression and its product augurin in the choroid plexus: impact on fetal brain development, cerebrospinal fluid homeostasis and neuroprogenitor cell response to CNS injury

<p>Abstract</p> <p>Background</p> <p>The content and composition of cerebrospinal fluid (CSF) is determined in large part by the choroid plexus (CP) and specifically, a specialized epithelial cell (CPe) layer that responds to, synthesizes, and transports peptide hormones into and out of CSF. Together with ventricular ependymal cells, these CPe relay homeostatic signals throughout the central nervous system (CNS) and regulate CSF hydrodynamics. One new candidate signal is augurin, a newly recognized 14 kDa protein that is encoded by <it>esophageal cancer related gene-4 </it>(<it>Ecrg4</it>), a putative tumor suppressor gene whose presence and function in normal tissues remains unexplored and enigmatic. The aim of this study was to explore whether <it>Ecrg4 </it>and its product augurin, can be implicated in CNS development and the response to CNS injury.</p> <p>Methods</p> <p><it>Ecrg4 </it>gene expression in CNS and peripheral tissues was studied by <it>in situ </it>hybridization and quantitative RT-PCR. Augurin, the protein encoded by <it>Ecrg4</it>, was detected by immunoblotting, immunohistochemistry and ELISA. The biological consequence of augurin over-expression was studied in a cortical stab model of rat CNS injury by intra-cerebro-ventricular injection of an adenovirus vector containing the <it>Ecrg4 </it>cDNA. The biological consequences of reduced augurin expression were evaluated by characterizing the CNS phenotype caused by <it>Ecrg4 </it>gene knockdown in developing zebrafish embryos.</p> <p>Results</p> <p>Gene expression and immunohistochemical analyses revealed that, the CP is a major source of <it>Ecrg4 </it>in the CNS and that <it>Ecrg4 </it>mRNA is predominantly localized to choroid plexus epithelial (CPe), ventricular and central canal cells of the spinal cord. After a stab injury into the brain however, both augurin staining and <it>Ecrg4 </it>gene expression decreased precipitously. If the loss of augurin was circumvented by over-expressing <it>Ecrg4 in vivo</it>, BrdU incorporation by cells in the subependymal zone decreased. Inversely, gene knockdown of <it>Ecrg4 </it>in developing zebrafish embryos caused increased proliferation of GFAP-positive cells and induced a dose-dependent hydrocephalus-like phenotype that could be rescued by co-injection of antisense morpholinos with <it>Ecrg4 </it>mRNA.</p> <p>Conclusion</p> <p>An unusually elevated expression of the <it>Ecrg4 </it>gene in the CP implies that its product, augurin, plays a role in CP-CSF-CNS function. The results are all consistent with a model whereby an injury-induced decrease in augurin dysinhibits target cells at the ependymal-subependymal interface. We speculate that the ability of CP and ependymal epithelium to alter the progenitor cell response to CNS injury may be mediated, in part by <it>Ecrg4</it>. If so, the canonic control of its promoter by DNA methylation may implicate epigenetic mechanisms in neuroprogenitor fate and function in the CNS.</p

Co-localization and regulation of basic fibroblast growth factor and arginine vasopressin in neuroendocrine cells of the rat and human brain

<p>Abstract</p> <p>Background</p> <p>Adult rat hypothalamo-pituitary axis and choroid plexus are rich in basic fibroblast growth factor (FGF2) which likely has a role in fluid homeostasis. Towards this end, we characterized the distribution and modulation of FGF2 in the human and rat central nervous system. To ascertain a functional link between arginine vasopressin (AVP) and FGF2, a rat model of chronic dehydration was used to test the hypothesis that FGF2 expression, like that of AVP, is altered by perturbed fluid balance.</p> <p>Methods</p> <p>Immunohistochemistry and confocal microscopy were used to examine the distribution of FGF2 and AVP neuropeptides in the normal human brain. In order to assess effects of chronic dehydration, Sprague-Dawley rats were water deprived for 3 days. AVP neuropeptide expression and changes in FGF2 distribution in the brain, neural lobe of the pituitary and kidney were assessed by immunohistochemistry, and western blotting (FGF2 isoforms).</p> <p>Results</p> <p>In human hypothalamus, FGF2 and AVP were co-localized in the cytoplasm of supraoptic and paraventricular magnocellular neurons and axonal processes. Immunoreactive FGF2 was associated with small granular structures distributed throughout neuronal cytoplasm. Neurohypophysial FGF2 immunostaining was found in axonal processes, pituicytes and Herring bodies. Following chronic dehydration in rats, there was substantially-enhanced FGF2 staining in basement membranes underlying blood vessels, pituicytes and other glia. This accompanied remodeling of extracellular matrix. Western blot data revealed that dehydration increased expression of the hypothalamic FGF2 isoforms of ca. 18, 23 and 24 kDa. In lateral ventricle choroid plexus of dehydrated rats, FGF2 expression was augmented in the epithelium (Ab773 as immunomarker) but reduced interstitially (Ab106 immunostaining).</p> <p>Conclusions</p> <p>Dehydration altered FGF2 expression patterns in AVP-containing magnocellular neurons and neurohypophysis, as well as in choroid plexus epithelium. This supports the involvement of centrally-synthesized FGF2, putatively coupled to that of AVP, in homeostatic mechanisms that regulate fluid balance.</p

Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation